The mammalian target of rapamycin (mTOR) is a serine/threonine kinase, which belongs to a large family of PI3K-related kinases, which also includes DNA-dependent protein kinase, ATM and ATR. Biochemical and genetic studies demonstrated that mTOR integrates growth factor stimulation, energy, and nutrient availability to regulate biosynthetic processes responsible for cell growth, size, and cell cycle progression. The regulation of mTOR kinase activity in response to various extracellular cues has been extensively studied in the last decade. A whole range of cellular proteins with various enzymatic activities, scaffold and adaptor functions have been implicated in signaling to and regulating the activity of mTOR, including protein kinases PKB/Akt and AMPK, tumor suppressors PTEN and tuberous sclerosis complex TSC1/2, small GTP-binding protein Rheb, scaffolding proteins Raptor and Rictor. In an activated state, mTOR transduces signaling and metabolic information by phosphorylating major downstream targets, such as S6Ks, 4E-BP1 and PKB/Akt. Studies from various laboratories indicated that mTOR exists in two functionally distinct complexes, termed mTOR complex 1 (mTORC1) and mTORC2. The mTORC1 contains the core components mTOR, raptor, and mLST8/GβL, and is sensitive to rapamycin. The mTORC2 is believed to be rapamycin insensitive and contains mTOR, Rictor, and mLST8/GβL. Rapamycin and its analogues are currently evaluated as anticancer drugs in a number of clinical trials. In addition, they are now widely used for coating stents to reduce post-stenting restenosis after coronary angioplasty. In contrast to yeasts, which have two TOR genes, mammals possess only one gene, known to encode a single polypeptide with molecular weight of approximately 280 kDa.
One important downstream target of mTOR is S6K. S6 kinase (p70 S6 kinase (p70S6k)) is responsible for S6 phosphorylation of the ribosomal S6 protein, which is a component of the 40S subunit of eukaryotic ribosomes (i.e., the cellular machinery responsible for translation of mRNA and protein synthesis). It is also believed to be the major physiological S6 kinase in mammalian cells (Proud, 1996 Trends Biochem. Sci. 21:181-185). The 40S ribosomal protein S6 is a component of the 40S subunit of eukaryotic ribosomes. The S6 protein is phosphorylated in response to certain cellular signaling events such as hormone or growth factor induced cellular proliferation.
S6 Kinase is activated by a variety of growth factors such as insulin, and mitogens (Alessi et al., 1998 Curr. Biol. 8:69-81). Certain drugs that regulate S6 kinase activity have been identified including rapamycin, which is the most potent inhibitor of S6 kinase (Pullen et al., 1997 FEBS Letters 410:78-82). The structure of and some functions of S6 kinase alpha and S6 kinase beta are disclosed in U.S. Pat. No. 6,830,909, herein incorporated by reference in its entirety.
S6 kinase has been shown to be both phosphorylated and acetylated. It was found that S6 kinase protein is acetylated both in vivo and in vitro by p300 and P/CAF acetyltransferases. More accurate to say that in conditions under which S6K acetylation is increased (i.e., presence of HDAC inhibitors/overexpression of p300), S6 kinase activity and 412 phosphorylation are reduced. Mutation of the P-loop lysines (which are acetylated by p300 in vitro) to glutamine, which mimics acetylation, results in the complete inactivation of S6Ks and loss of 412 phosphorylation. S6K2 (S6K beta) has also been shown to possesses an AT-hook DNA binding motif. Thus, S6 kinase 2 protein binds to DNA and is thereby activated which in turn stimulates its kinase activity. Therefore, S6K2 is thought to transduce growth-promoting effects in response to mitogens and nutrients. This may involve the regulation of transcription factors and/or chromatin remodeling proteins by phosphorylation, when S6K2 complexes with DNA and is activated by this interaction. Regulation of S6 Kinase protein activity and related methods are disclosed in WO 2007/019421, incorporated herein by reference in its entirety.